The molecular mechanism of Aire: partnering with DNA-PK

描述(申请人提供)：自身免疫性疾病困扰着7-10%的美国人，其发病率还在上升。APECED患者具有多器官自身免疫性疾病，对其的研究对免疫耐受和自身免疫性疾病的研究具有重要意义。APECED背后的突变基因编码Aire，这是一种具有转录调节因子特征的几个结构域的大蛋白。编码小鼠Aire基因的突变也可导致多器官自身免疫。对这一模型的研究表明，AIRE主要通过诱导大量外周组织抗原(PTA)的表达，在罕见的胸腺基质细胞群体中发挥作用。因此，当分化的T细胞通过胸腺渗透时，那些能够对这种PTA产生反应的T细胞可以避免克隆缺失；当它们出现在外周并遇到同源抗原时，就会产生自身免疫。艾尔的分子机制仍然是个谜。它控制着基因的表达，但似乎不像传统的转录因子那样起作用。相反，它似乎在其活动中高度合作，参与包含不同功能的蛋白质的大型多蛋白复合体，参与核运输、染色质结合/结构、转录调控或前mRNA加工。我们的初步研究强调了一个意想不到的Aire伙伴：DNA依赖蛋白激酶(DNA-PK)，通常与DNA双链断裂(DSB)的修复通过非同源末端连接有关，但最近也参与了转录延长的控制。这个被提议的项目的目标是阐明Aire和DNA-PK如何相互作用来促进一大部分基因组的转录，特别是在罕见的胸腺基质细胞中。建议的研究旨在：1)从结构和功能上定义含有[Aire/DNA-PK]的复合体，结合顺序亲和纯化、候选伙伴共沉淀、细胞中的shRNA敲除和基因敲除小鼠。2)确定Aire是否促进DNADSB的生成/稳定性，追寻(来自我们初步研究的)假设，即它的作用类似于抗癌药物依托泊苷，以抑制DNA拓扑异构酶-2在引入DNA切割以缓解与转录相关的扭转应力后分解DNA切割。3)确定Aire是否通过组蛋白“驱逐”机制对转录产生影响，检验这一假设，即随着转录机制沿DNA的进展，Aire促进负责分解和重组核小体的复合体(包括DNA-PK、PARP1、TOP2、FACT、H2AX)的招募和/或有效性。这些研究应该会对Aire控制免疫耐受的分子机制产生新的见解。除了APECED，这种T细胞耐受模式被认为在常见的自身免疫性疾病中发挥着重要作用，特别是1型糖尿病和重症肌无力。用抗癌药物、依托泊苷或相关药物成功干预Aire基因缺陷小鼠的自身免疫性疾病，将代表着这一耐受途径可以被靶向治疗的原则证明。

DESCRIPTION (provided by applicant): Autoimmune diseases afflict 7-10% of Americans, and their incidence is rising. APECED patients have a multi-organ autoimmune disorder, the study of which has yielded important insights into immunological tolerance and autoimmune diseases more generally. The mutated gene underlying APECED encodes Aire, a large protein with several structural domains characteristic of a transcriptional regulator. Mutation of the locus encoding murine Aire also resulted in multi-organ autoimmunity. Studies on this model revealed Aire to operate primarily within a rare population of thymic stromal cells by inducing the expression of a large repertoire of peripheral-tissue antigens, or PTAs. Consequently, as differentiating T cells percolate through the thymus, those capable of responding to such PTAs avoid clonal deletion; when they emerge into the periphery and encounter cognate antigen, autoimmunity ensues. Aire's molecular mechanisms remain enigmatic. It controls gene expression but does not appear to act as a conventional transcription factor. Rather, it seems to be highly cooperative in its activities, participating in large multi-protein complexes that incorporate proteins of diverse function, involved in nuclear transport, chromatin binding/structure, transcriptional regulation or pre-mRNA processing. Our preliminary studies highlighted an unexpected Aire partner: DNA-dependent protein kinase (DNA-PK), usually associated with the repair of DNA double-stranded breaks (DSBs) via non-homologous end joining, but also recently implicated in the control of transcriptional elongation. The goal of this proposed project is to elucidate how Aire and DNA-PK interact to promote transcription of a large, but select, portion of the genome specifically in rare thymic stromal cells. Proposed studies aim to: 1) Structurally and functionally define the [Aire/DNA-PK]-containing complex(es), combining sequential affinity-purifications, candidate partner co-precipitations, shRNA knockdowns in cells, and gene-knockout mice. 2) Determine whether Aire promotes the generation/stability of DNA DSBs, pursuing the hypothesis (issuing from our preliminary studies) that it operates like the anti-cancer drug etoposide to inhibit DNA topoisomerase-2 from resolving DNA cuts after introducing them to relieve the torsional stress associated with transcription. 3) Determine whether Aire impacts on transcription via the histone "eviction" machinery, testing the hypothesis, that it promotes the recruitment and/or effectiveness of a complex (including DNA-PK, PARP1, TOP2, FACT, H2AX) responsible for disassembling and re-assembling nucleosomes as the transcriptional machinery progresses along DNA. These studies should yield new insights into the molecular mechanisms by which Aire controls immunological tolerance. Besides APECED, this mode of T cell tolerization is thought to play an important role in common autoimmune diseases, notably type-1 diabetes and myasthenia gravis. Successful intervention in the autoimmune disease of Aire-deficient mice with the cancer drug, etoposide, or allied drugs would represent proof-of-principle that this tolerance pathway can be targeted for therapeutic benefit.